Non-infectious HIV particles lacking long terminal repeats

ABSTRACT

An immunogenic HIV retrovirus-like particle which is non-infectious and non-replicating and which is useful as a candidate vaccine component against HIV infection, is produced by genetic engineering. A DNA molecule comprising the HIV genome devoid of long terminal repeats is incorporated into an expression vector, which is introduced into mammalian cells for expression of the HIV retrovirus-like particle.

FIELD OF INVENTION

The present invention relates to the preparation of humanretrovirus-like particles, specifically HIV-like particles, which areimmunogenic and non-infectious. These preparations will serve ascandidates for whole-virus-like vaccines for human retrovirus diseasesand should not be subject to the ethical concerns regarding theproduction of classical whole-virus vaccines from infectious viruspreparations.

BACKGROUND TO THE INVENTION

Among all diseases caused by retroviruses in humans and animals, theacquired immunodeficiency syndrome (AIDS) and the adult T-cellleukemia-lymphoma (ATLL) represent the most dramatic human diseases dueto HIV and HTLV-1 retroviruses, respectively. The etiologic agent of theacquired immune deficiency syndrome (AIDS) is a human retrovirus termedhuman immunodeficiency virus (HIV) of which there are presently twomajor subgroups, HIV-1 and HIV-2. These viruses are responsible for anever widening world-wide epidemic of immune deficiency and centralnervous system (CNS) disorders characterized by a slow, yet progressive,degeneration of immune and CNS functions. HIV-1 affects mainly NorthAmerica, Western Europe, Haiti and Central Africa while HIV-2 is foundpredominantly in West African countries. The earliest symptoms of HIVinfection include an acute influenza-like syndrome which persists for 2to 3 weeks. Several weeks to many months or years following infection,lymphadenopathy and/or progressive depletion in CD4⁺ T-helperlymphocytes becomes apparent and disease evolves to the point whereimmune deficiency becomes manifest. The diagnosis of HIV infection isconfirmed by laboratory tests which include the detection ofHIV-specific antibodies and/or HIV antigens in patient sera, and theisolation of infectious virus from patients body fluids or cells. Asimilar disease is observed in rhesus macaques infected with the simianacquired immunodeficiency virus (SIV).

Immune deficiency in HIV infection is characterized by opportunisticinfections with microbial agents which are not normally associated withdisease in otherwise healthy individuals. The severity of theseinfections is exacerbated by the loss of helper T-cell function, which,when combined with other symptoms, such as diarrhoea and weight loss,leads to a general wasting syndrome. Death usually results from one ormore opportunistic infections. As mentioned above, CNS involvement isanother manifestation of AIDS and can be the result of directHIV-induced neurological disease as well as that of opportunisticinfections.

The predominant host cells for HIV in infected individuals are the CD4⁺T-helper cell and the monocyte/macrophage. However, more and moreevidence points to the fact that HIV can infect a wide variety of celltypes, CD4⁺ and CD4⁻, both in vivo and in vitro. These cell typesinclude those of the haematopoietic system, the central nervous system,the gastrointestinal tract, and skin. This wide host cell tropism mostlikely accounts for the plethora of symptoms and the severity of diseaseassociated with HIV infection.

HIV-1 and 2 have been the subject of massive and unprecedented researchefforts in recent years in a number of areas including vaccinestrategies. The development of an efficacious vaccine for prevention ofHIV infection, is of considerable importance as it can be easilyrecognized that prevention of infection is the best way to combat anyinfectious disease.

Various strategies are currently being used in attempts to develop aneffective vaccine against AIDS. Some of these strategies are brieflyoutlined along with their respective advantages and disadvantages.

A subunit HIV vaccine consists of one or more purified HIV immunogens,either obtained from disrupted whole virus or produced in geneticallyengineered eukaryotic or bacterial expression systems. An importantadvantage of this type of vaccine is the relative ease with which theseproducts can be produced. However, this advantage can be countered bythe fact that subunit vaccines only contain a subset of HIV antigenicdeterminants, which in some cases can lead to a less than optimal immuneresponse. Moreover, viral protein subunits may adopt different spatialconformations when extracted from the context of the whole-virusparticle. This may affect the structure of important conformationalepitopes and result in inefficient immune responses.

Live recombinant virus vaccines consist of a non-pathogenic virus, suchas vaccinia or adenovirus, which has one or more non-essential genesreplaced by a nucleotide sequence encoding one or more HIV antigens.Live recombinant viruses can often induce efficient immune responses tosingle subunits of a particular pathogenic virus. However, as withsubunit vaccines, recombinant virus vaccines express only a fraction ofthe total antigens of a given virus which can be disadvantageous whenhighly efficient immune responses are required.

Future vaccines may consist of synthetic peptides containing multipleepitopes of a given pathogen. These peptides, coupled to a carrierprotein and combined with an appropriate adjuvant, are potentiallycapable of eliciting good and lasting humoral and cellular immuneresponses against multiple components of a pathogen. The development ofan efficacious synthetic peptide vaccine for AIDS is likely to requirethe full identification of all the functionally important immunologicaldeterminants of HIV-1 and HIV-2, a task which may not be completed inthe very near future. An important disadvantage of peptide vaccines isthe difficulty to produce synthetic molecules mimicking conformationalepitopes (immunological determinants which are formed by distant aminoacid residues brought together in space by protein folding). Ifconformational epitopes are important for protection against aparticular infectious agent, it is unlikely that traditional peptidevaccine designs will prove successful.

Inactivated, whole-virus vaccines consist of a purified preparation ofintact particles from a given viral pathogen which has been renderednon-infectious by chemical or physical means. The inherent advantages ofthese vaccines are their relative ease of production and the fact thatall or most of the important immunological epitopes of the virus arepresent. However, a major disadvantage of these vaccines is thatinfectious virus must be propagated on a large scale, thereby exposingproduction workers to significant risks, depending on the nature of thepathogen. Equally important is the fact that the virus must be renderedcompletely non-infectious. This poses ethical problems since it isextremely difficult to demonstrate that all infectious genetic materialhas been removed. Moreover, extensive inactivation regimes to kill allinfectious viruses are likely to destroy or alter various immunologicalepitopes, thereby compromising the immunogenicity of the vaccine.

This invention describes a method to produce non-infectious,retrovirus-like particles as the basis for a candidate human vaccineagainst AIDS. This invention is also applicable to the production ofsimilar particles serving as a candidate vaccine to other retrovirusdiseases which include, but are not limited to, the simian acquiredimmune deficiency syndrome caused by the simian immunodeficiency virus(SIV), and certain forms of human T-cell leukemia-lymphoma caused by thehuman T-cell leukemia virus I (HTLV-1).

A non-infectious retrovirus-like particle of this nature does notcontain any infectious RNA and has the advantage of including all orpart of the major viral antigens in their native configuration. Theproduction of such a particle does not obligatorily require physical orchemical inactivation, thus avoiding the possible destruction ofimportant immunological determinants. The ability of this particle toelicit potent immune responses to native viral proteins without any riskof inducing infection and disease makes it an important new candidatevaccine for evaluation in animal models and humans.

SUMMARY OF INVENTION

This invention describes a general method for the production of humanretrovirus-like particles, specifically HIV-like particles, which areimmunogenic and non-infectious. This preparation of geneticallyengineered HIV-like particles will serve as a candidate "whole-virus"vaccine for AIDS and should not be subject to the specific ethicalconcerns regarding the production of classical whole-virus vaccines frominfectious virus preparations. It should be noted that the methodologiesdeveloped here for the AIDS virus are directly applicable to all humanand non-human retroviruses and can be used to produce any retroviralcandidate vaccine, including, but not limited to, the AIDS virus, andnot limited to human pathogens. Furthermore, the same methodologies canbe used to produce non-infectious retroviral particles to serve asantigens in diagnostic immunoassays for retroviral diseases. For thepurpose of clarity, the discussion here pertains to actual examplesemploying the AIDS virus, but it is to be assumed that this inventioncovers all retroviruses.

The present invention describes the engineering of cultured cells toproduce retroviral proteins which self-assemble into virus-likeparticles in the absence of the production of an infectious retrovirusgenomic RNA molecule. A virus-like particle can be defined here as adefective virion which is incapable of infecting a host cell due to thepresence of one or more genetic modifications of viral genes or othergenetic elements which are functionally critical at some stage of thevirus life cycle. Virus-like particles may or may not contain all of theviral proteins normally found in infectious virions and may or may notcontain RNA. If RNA is contained within the particle, it will beincapable of infecting a host cell.

In the present examples pertaining to HIV, the production ofnon-infectious virus-like particles required the isolation of a DNAfragment from the HIV provirus containing relevant protein codinginformation and deficient in genomic elements required for replicationand transcription of the retrovirus genome. This DNA fragment was linkedto a heterologous promoter and transfected into a cultured cell line toallow for the expression of HIV-1 proteins and their assembly intovirus-like particles. The unique features of this invention aredescribed in detail below but centre on the fact that the presentinvention consists of a preparation of virus-like particles, which willnot require chemical inactivation, and which can serve as a candidatevaccine for retroviral pathogens. Moreover, the method allows for theproduction of non-infectious virus-like particles containingmodifications in one or more viral proteins so as to enhance theimmunogenicity of the particles. Thus, the resultant candidate vaccinewill represent a safe preparation of virus-like particles containing anoptimized set of immunological epitopes necessary for stimulating apotent immune response.

The safety of genetically engineered, non-infectious HIV-like particlescan be guaranteed by the genetic engineering steps employed to producethese particles. Viral genetic elements required for replication areeliminated and a variety of genetic modifications can be introduced inthe viral DNA sequences to be inserted into the producing cell line.These mutations can affect gene functions, gene products or geneticelements required for viral infectivity but not involved in thesynthesis of the major viral proteins required for particle assembly orimmunogenicity. With this strategy in hand, it becomes apparent to thoseskilled in the art that expression vectors employed to produce suchnon-infectious particles will not yield infectious viruses.

The techniques for producing a preparation of safe, non-infectiousvirus-like particles stem from knowledge which is available to thoseskilled in the art, but which has been applied here in a unique fashionto produce whole virus-like particles devoid of infectious HIV viral RNAor RNA which can be replicated into double-stranded DNA in a recipientcell. We have developed an expression system for HIV antigens whichresults in the production of the major HIV-1 antigens in an engineeredcell in the absence of the production of an infectious genomic RNAmolecule. We have demonstrated co-expression of the envelope and coreantigens and provide evidence that these products are assembled intoretrovirus-like particles and can be purified by means employed topurify normal, infectious preparations of HIV particles. In addition,these purified virus-like particles induce efficient HIV-1 specificantibody responses in immunized mice and cross-reactivity with other HIVstrains, including HIV-2.

It is important to note that HIV-like particles and the cell lines usedto produce these particles described here are significantly differentfrom prior art retroviral studies involving the insertion of retrovirusgenomes into cultured cell lines. Prior art experiments involved theconstruction of cell lines expressing murine retroviral proteins capableof packaging defective retroviral genomes carrying foreign genes. Thepackaging of defective retroviral genomic RNA molecules carrying foreigngenes into virus particles allows for the efficient introduction ofthese RNAs into recipient cells. Once transduced into recipient cells,these recombinant viral RNAs become reverse transcribed intodouble-stranded DNA and integrated into the chromosomes of the cell,thereby genetically transforming the recipient cell. In the inventiondescribed here, the use of HIV-1 expression vectors devoid of longterminal repeat (LTR) elements results in the production ofnon-infectious virus-like particles which are intended for use as avaccine and not for the transduction of recombinant retroviral RNAmolecules into recipient cells for the purpose of genetictransformation. Indeed, in the present invention, RNA packaging intovirus particles can be minimized and any RNA which is packaged cannotundergo the process of reverse transcription. Therefore, the presentinvention differs from prior art studies of retrovirus proteinexpression in the intended uses, methods employed, and the nature andcharacteristics of the resultant products.

As indicated above, the final products of the present invention arepreparations of virus-like particles which are non-infectious due to theabsence of an infectious retrovirus genome within the particles.Moreover, the present invention allows for genetic manipulations tooptimize the immunogenicity of the particles and vaccine efficiency inimmunized recipients. It is envisioned that significant alterations canbe made to certain viral protein components of the geneticallyengineered particles through alterations in the DNA sequences encodingthese components. These alterations are likely to involve the insertionof extra copies of various important immunological epitopes into virusprotein regions, which are not critical for particle assembly, togenerate candidate multivalent chimeric vaccines. Additional alterationsmight involve the deletion of certain protein regions which may beimmunosuppressive or lead to the production of autoimmune disorders orenhancing antibodies. The alterations would be designed so as to avoidinterference with particle assembly and create modified particlescapable of eliciting an optimized immune response. The invention alsoallows for the production of hybrid viral particles containing antigensfrom multiple strains of a given infectious virus, or even fromdifferent viruses altogether.

In summary, an invention is described which entails the expression ofnormal or modified retroviral proteins in a cultured cell line for theintended purpose of producing non-infectious, assembled virus-likeparticles as a candidate vaccine. The invention describes the use ofstably engineered cell lines using inducible and constitutive promotersand an example of one method to produce such particles. It is not to be.assumed that the genetic engineering examples described here are theonly means of expressing non-infectious retrovirus-like particles.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a genetic map of the provirus of HIV strain LAV-1_(BRU)which indicates the relative locations of the long terminal repeats(LTRs), the major protein coding sequences (gag, pol, and env) and theremaining open reading frames which encode minor structural and/orregulatory proteins (vif, rev, vpu, vpr, tat, and nef);

FIG. 2 shows a map of the HIV expression construct pHIV-SV whichcontains an 8.3 kb SacI-XhoI fragment from the LAV-1_(BRU) genome. Thisfragment, which lacks LTR elements, was inserted into an expressionvector containing the SV40 virus early promoter and late polyadenylationsite;

FIG. 3 shows a map of the HIV expression construct pHIV-CHO-SV whichcontains an 8.3 kb SacI-XhoI fragment from the LAV-1_(BRU) genome. Thisfragment, which lacks LTR elements, was inserted into an expressionvector containing the adenovirus major late promoter, two copies of thehuman cytomegalovirus immediate early enhancer, the SV40 virus latepolyadenylation site, and the SV40 virus origin or replication. Thelatter element was inserted to allow for transient expression in monkeyCOS cells;

FIG. 4 shows a sucrose density gradient centrifugation profile ofHIV-1-like particles expressed in transiently transfected COS cells.Fractions containing virus-like particles were identified by RT assayand the density of individual fractions was determined gravimetrically.The peak of RT activity was found in the gradient fraction whichexhibited a density of approximately 1.16 g/mL;

FIG. 5 shows a map of the HIV expression plasmid pHIV-Ad. This vector issimilar to pHIV-CHO-SV shown in FIG. 3 but lacks the SV40 virus originof DNA replication and was intended for stable transfection studies;

FIG. 6 shows a sucrose density gradient centrifugation profile of HIV-1like particles expressed in stably transfected COS cells. Fractionscontaining virus-like particles were identified by RT assay;

FIG. 7 represents the titration curves of IgG antibody reactivitiesagainst four recombinant HIV-1 antigens (gp120, gp160, gp41 and p24), asmeasured by antigen-specific EIA's. Each point is the mean of twodeterminations obtained independently for anti-sera collected from twomice immunized with two injections of virus-like particles. Absorbancevalues obtained for normal mouse serum used as a control were alwaysless than 0.1;

FIG. 8 represents the titration curves of IgG antibody reactivitiesagainst five different recombinant HIV-1 antigens (HIV-1 gp120, gp160,gp41, p24 and HIV-2 gp120), as measured by antigen-specific EIA's. Eachpoint is the mean of two determinations obtained independently forantisera collected from two mice immunized with three injections ofHIV-like particles. Absorbance values obtained for normal mouse serumused as a control were always less than 0.1;

FIG. 9 represents the titration curves of IgG antibody reactivitiesagainst three recombinant HIV antigens (HIV-1 gp160 and gp120, HIV-2gp120), as measured by antigen specific EIA's. Each point is the mean oftwo determinations obtained independently for antisera from two miceimmunized with three injections of HIV-1-like particles. In contrast toFIG. 8, the strongest immune response was observed with HIV-1 rgp160,which was obtained from a manufacturer (Transgene) different from theone who supplied the rgp160 (Repligen) used in FIG. 8. Absorbance valuesobtained for normal mouse serum were always less than 0.1;

FIG. 10 shows a map of the HIV expression plasmid pMT-HIV which issimilar to the pHIV-SV vector shown in FIG. 2. pMT-HIV contains thehuman metallothionein IIa promoter in place of the SV40 virus earlypromoter used in pHIV-SV;

FIG. 11 depicts the distribution of RT activity and p24 antigenassociated with high molecular weight material present in thesupernatants of stably transfected Vero cells over a period of eightdays. The cells were grown to confluence and 5 uM CdCl₂ was added tostimulate virus-like particle production. Every twenty four hours theculture medium was replaced with fresh medium containing 5 uM CdCl₂ andthis process was repeated each day for eight days. After collection ofthe day 8 sample, RT and p24 assays were performed on all samplessimultaneously; and

FIG. 12 shows the sucrose density gradient centrifugation profiles forHIV-1-like particles prepared from two lines of stably transfected COScells. COS-HIV-3 was derived by transfection of COS cells with thepHIV-Ad expression vector and COS-HIV-6 was derived by transfection witha similar vector containing a 26 bp deletion in the RNA packaging signalsequence.

GENERAL DESCRIPTION OF THE INVENTION

FIG. 1 shows a functional map of the HIV-1 provirus indicating thelocation of various genes as well as the long terminal repeat (LTR)elements which are required for retroviral gene expression and genomereplication. In the present invention, we have demonstrated that it waspossible to produce HIV-1-like particles in transiently transfectedmonkey COS cells by inserting an 8.3 Kb SacI-XhoI DNA fragment from theprovirus of LAV-1_(BRU) into a simple eukaryotic expression vectoremploying the SV40 virus early promoter (FIG. 2). The proviral fragmentemployed contained the viral protein coding information found betweenthe LTR elements and, therefore, lacked the genetic elements necessaryfor the reverse transcription of any RNA molecule transcribed from thisfragment. Upon transfection of this vector into COS cells, HIV-1 proteinexpression was confirmed by metabolic labelling and immuneprecipitation, by Western blot analysis of pelleted material using aspecific anti-HIV antiserum and detection of reverse transcriptase (RT)activity in the supernatant of transfected cells. Evidence for theformation of HIV-like particles was obtained by sucrose density gradientcentrifugation of the high molecular weight material released in thesupernatant of transfected cells. In this experiment, RT activity wasshown to band at a density similar to that of intact retrovirusparticles. It was further established that SIV-like particles could beengineered using the same method.

To establish stably engineered cell lines for more efficient productionof non-infectious HIV-1-like particles, HIV-1 protein coding informationwas inserted into an expression vector employing the adenovirus majorlate promoter and transfected into COS cell along with a plasmidspecifying resistance to the antibiotic G418 (FIG. 3). After examinationof a number of G418 resistant cell clones, several were identified whichconstitutively produced high molecular weight material containing RTactivity in association with major HIV-1 antigens. That actualvirus-like particles were being produced was demonstrated by sucrosedensity gradient centrifugation and electron microscopic analysis. Thelatter revealed the presence of virus-like particles budding from theplasma membrane of transfected cells.

To demonstrate the potential for non-infectious HIV-1-like particles tofunction as a candidate vaccine, mice were immunized with a preparationof non-infectious HIV-1-like particles produced by stably transfectedCOS cells. In these experiments, particles were purified bysedimentation and sucrose banding and used as immunogens. After twoinjections, an efficient antibody response against various HIV-1proteins and peptides was observed and virus neutralizing activity wasdetected for two HIV-1 strains. After a third injection, HIV-1 specificantibodies showed cross-reactivity with the envelope glycoprotein ofHIV-2.

To improve the levels of non-infectious HIV-1 particle production inengineered cell lines, an expression vector containing the humanmetallothionein IIa promoter and the HIV-1 protein coding DNA fragmentwas developed to allow for the inducible expression of virus-likeparticles in stably engineered cell lines (FIG. 10). Afterco-transfecting this vector into Vero cells along with the G418resistance marker, numerous clones were identified which produced highlevels of particulate RT activity. HIV-like particle production wasverified by electron microscopy and p24 antigen assays indicated thatlevels of particle production were higher than normally observed in aninfection of human leukaemic T-cells with infectious HIV-1. Moreover,particle production in the stably transfected Vero cells was maintainedfor at least eight consecutive days after reaching confluence when thecells were given fresh medium and cadmium chloride every twenty fourhours. Inducible and long-term expression was not limited to monkey Verocells. Metal-responsive expression of substantial amounts of particleswas also observed in a human colon adenocarcinoma cell line. These dataindicate that a wide variety of cell lines are suitable for thelarge-scale production of non-infectious virus-like particles.

Although one major advantage of this method is the possibility toproduce virus-like particles which may potentially contain all of theviral proteins in their native configuration, it is neverthelesspossible to produce modified virus-like particles containing viralantigens with altered structures. It may be determined that theintroduction of epitopes from alternative viral strains into certainregions of the HIV-1 envelope glycoprotein may enhance itsimmunogenicity and is of net benefit in spite of potential negativeeffects associated with the possible alteration of native conformationalepitopes. In addition, it may be possible to improve the immunogenicityof HIV-like particles by deleting certain enhancing or immunosuppressiveregions of the envelope or other viral proteins. To demonstrate thefeasibility of producing virus-like particles containing modifiedenvelope proteins, we first mutated the proteolytic processing site ofthe gp160 envelope glycoprotein precursor to block processing of thegp160 molecule into the mature gp120 envelope glycoprotein and the gp41transmembrane glycoprotein. This experiment was performed to determineif unprocessed gp160 could be targeted to the membrane of virus-likeparticles. Since modified gp120 molecules may be easily shed fromvirus-like particles as a result of a reduced affinity for the gp41transmembrane glycoprotein, it may be necessary to block proteolyticprocessing to efficiently retain modified envelope molecules on theparticles. Sucrose density gradient analysis of particles produced froman expression construct containing a mutation in the region coding forthe processing site revealed the presence of uncleaved gp160 tightlyassociated with RT activity in heavy molecular weight material releasedfrom transfected cells. Furthermore, additional expression constructsencoding envelope glycoproteins with epitope insertions at defined sitesdemonstrated the feasibility of producing particles with modified gp120envelope glycoproteins containing specific aminoacid insertions.Conversely, deletion of DNA sequences coding for the major part of gp120resulted in the production of virus-like particles lacking theextracellular domain of the envelope protein.

Genetically engineered, non-infectious HIV-like particles are designedto represent a completely safe candidate vaccine for use in humans. Thisis due to the fact that cell lines producing the virus-like particles donot contain the genetic elements required for reverse transcription.However, with the elimination of only the LTR elements, it may be arguedthat recombination of the protein coding sequences with heterologous LTRelements in either the vaccine-producing cell or in the vaccinerecipient may result in the regeneration of an infectious retrovirus.This possibility can be completely eliminated by making a number ofadditional genetic modifications in regions of the HIV nucleotidesequences which are necessary for infectivity but dispensable forparticle production and immunogenicity. Such regions include the RNApackaging signal, the region coding for the C-terminus of the gag p15product, and the vif, integrase, reverse transcriptase, and tat genes.An expression construct containing mutations of this nature in additionto the deletion of the LTR elements could not give rise to infectiousretrovirus, even if recombined with functional LTR elements. In thepresent invention, we have demonstrated that it was possible to delete26 nucleotides between the first splice donor and the initiation codonfor gag which encompasses part or all of the HIV-1 RNA packaging signal.In these experiments, virus-like particles expressed by this constructin stably transfected COS cells were indistinguishable from particlesexpressed from earlier constructs indicating that RNA packaging is not acritical function in the expression of non-infectious HIV-1 virus-likeparticles. Furthermore, we were able to show that the deletion in boththe Integrase and the Vif genes did not affect particle formation.

EXAMPLES Example 1

This Example illustrates the expression of the major HIV proteinantigens in cells transfected with an HIV expression construct.

FIG. 2 shows a diagram of the expression construct termed pHIV-SV whichcontains a single DNA fragment containing HIV-1 protein codinginformation from the LAV-1_(BRU) strain starting at nucleotide position678 and ending at nucleotide position 8944. This 8.3 kb fragment wasmade blunt-ended using the Klenow fragment of E. coli DNA polymerase Iand inserted into a blunt HindIII site of a Bluescript-based expressionvector containing the SV40 virus early promoter and late polyadenylationsite. The SV40 promoter and polyadenylation sites were obtained fromcommercially available cloning vectors. The pHIV-SV vector is suitablefor transient expression in monkey COS cells due to the presence of theSV40 virus origin of DNA replication.

Fifteen micrograms of the plasmid pHIV-SV was transfected into COS-7cells in a 25 cm² flask using standard calcium phosphate transfectionconditions. COS cells were maintained in Dulbecco's modified Eagle'smedium containing 10% fetal bovine serum. HIV protein expression wasanalyzed by ³ H-leucine metabolic labelling and immune precipitation ofintracellular and extracellular antigens. Twenty four hourspost-transfection, the cells were labelled with ³ H-leucine for 15 hoursafter which the medium was harvested and kept at 0° C. Cells were lysedby the addition of 1 mL of NP-40 lysis buffer (20 mM Tris-HCl, 150 mMNaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mMphenylmethylsulfonyl fluoride, pH 7.5) and cell lysates were held at 0°C. Supernatants and lysed cell samples were first reacted with normalhuman serum to clear any proteins non-specifically reacting with normalhuman immunoglobulins. These complexes were removed by binding toprotein-A agarose. The cleared medium and cell lysate samples weresubsequently reacted with a commercial human anti-HIV antiserum.HIV-specific immune complexes were isolated by binding to protein-Aagarose and the bound material was subjected to SDS PAGE. Gelelectrophoresis and fluorography demonstrated the expression of specificbands consistent with the p17, p24, gp41, p55, gp120 and gp160 productsof HIV-1. These bands were not present in control samples from cellswhich were not transfected with pHIV-SV.

Example 2

This Example illustrates the sedimentation of high molecular weightmaterial associated with reverse transcriptase activity from culturesupernatants of transfected COS cells.

Plasmid pHIV-CHO-SV (FIG. 3) is similar to pHIV-SV but employs theadenovirus major late promoter and two copies of the humancytomegalovirus immediate early enhancer instead of the SV40 virus earlypromoter. This plasmid, pHIV-CHO-SV, has been deposited with theAmerican Type Culture Collection, Rockville, Md., on Jul. 14, 1994 underAccession No. 75828. This plasmid also contains the SV40 virus origin ofreplication for transient expression analysis. The two copies of thehuman cytomegalovirus immediate early enhancer span the region fromnucleotide position -524 to -218 which was constructed from overlappingsynthetic oligonucleotides. The enhancer fragments are located upstreamfrom the adenovirus major late promoter which is contained within a 292bp XhoI-PvuII fragment from adenovirus-2 genomic DNA encompassing themajor late promoter and the 5' end of the first exon of the tripartiteleader. This 292 bp fragment was ligated to a synthetic 140 bp fragmentcontaining the 3' end of the first leader exon, all of the second leaderexon, and two thirds of the third leader exon, in a pre-splicedconfiguration.

Three 75 cm² flasks of COS-7 cells were each transfected with 35micrograms of the plasmid pHIV-CHO-SV and 30 mL of the 75 mL mediumsupernatant was collected three days post-transfection. High molecularweight complexes were pelleted through a 20% glycerol cushion containing50 mM Tris-HCl and 0.1 mM KCl, pH 7.8 in an SW28 centrifuge tube.Centrifugation was performed at 100,000×g for 90 minutes at 4° C.Samples from mock-transfected COS cells were included as controls. Thepellet was resuspended in 30 uL of Triton X-100 lysis buffer (50 mMTris-HCl, 100 mM NaCl, 1 mM dithiothreitol, 0.1% Triton X-100, pH 7.8)for subsequent reverse transcriptase activity analysis. One third of theresuspended sample was added to a 90 uL reaction mixture containing 40mM Tris-HCl, 4 mM dithiothreitol, 45 mM KCl, 10 mM MgCl₂, 20 uCi ³H-dTTP (80 Ci/mmol), 50 ug poly rA, and 1 ug oligo dT at pH 7.8. Thismixture was incubated at 37° C. for 30 minutes. Radioactiveincorporation into trichloroacetic acid-precipitable nucleic acidsindicated the presence of reverse transcriptase activity. The resultspresented in the following Table I show the radioactivity incorporatedfor each sample:

                  TABLE I                                                         ______________________________________                                        Sample              CPM incorporated                                          ______________________________________                                        Blank               14,110                                                    Mock transfected    18,053                                                    Transfected         390,220                                                   Purified MuLV reverse transcriptase                                                               5,416,920                                                 ______________________________________                                    

These data demonstrate that reverse transcriptase activity is present ina high molecular weight material released in the culture supernatants ofCOS cells transfected with an HIV expression plasmid.

Example 3

This Example illustrates the presence of multiple HIV-1 antigensassociated with high molecular weight material released in thesupernatants of transfected COS cells.

Twenty five ug of the plasmid pHIV-SV was transfected into COS cells ina 75 cm² culture flask using a commercial Lipofectin transfection kit.Eight mL of the 10 mL medium supernatant was harvested three dayspost-transfection and particulate material was pelleted at 100,000×gthrough a 20% glycerol cushion for subsequent Western blot analysis.Pellets were suspended in 100 uL of TNE (150 mM NaCl, 50 mM Tris-HCL, 1mM EDTA, pH 7.5) prior to the addition of SDS-PAGE sample buffer andelectrophoresed on a 12.5% SDS-polyacrylamide gels using standardmethodologies. Proteins were electrophoretically transferred toImmobilon membranes (Millipore) for subsequent reaction with a cocktailof four monoclonal antibodies specific for gp120 (DuPont, NEA-9384), p24(Dupont, NEA-9306), p17 (DuPont, NEA-9282) and gp41 (DuPont, NEA-9303),respectively. The second antibody was a goat anti-mouse IgG antibodyconjugated to alkaline phosphatase (Promega). Antibody reactions wereperformed in 5% Carnation instant milk in PBS and developed using analkaline phosphatase substrate solution (Bethesda ResearchLaboratories). Bands corresponding to all four HIV-1 products werepresent in the transfected cell samples but not observed with a controlsample from mock-transfected cells. The identity of the gp41 and gp120products was confirmed using single monoclonal antibodies in subsequentexperiments.

Example 4

This Example illustrates the buoyant density analysis of HIV-likeparticles produced from transfected COS cells.

Seventy micrograms of pHIV-CHO-SV were transfected into each of five 150cm² flasks of COS cells using the calcium phosphate method and culturesupernatants were harvested three days post-transfection. HIV-likeparticles were precipitated from growth medium by the addition of NaClto 0.15M and PEG-8000 to 9.3%, and layered on an 11 mL 20-60% sucrosedensity gradient in 100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4. Thegradient was centrifuged for 15 hours at 35,000 rpm in an SW40 rotor at4° C. The gradient was subsequently fractionated and aliquots fromindividual fractions were assayed for reverse transcriptase activityafter the addition of Triton X-100 to 0.2% and KCl to 0.25 mM to disruptvirus-like particles. A single peak of reverse transcriptase activitybanded at a buoyant density of approximately 1.16 g/mL consistent withthe production of retrovirus-like particles (FIG. 4). The density ofindividual fractions was measured gravimetrically to determine thedensity of the peak fraction.

Example 5

This Example illustrates the ability to generate stably transfected COScell clones expressing high molecular weight material containing reversetranscriptase activity.

The HIV expression plasmid pHIV-Ad (FIG. 5) is similar to pHIV-CHO-SVbut does not contain the SV40 virus origin of replication which wouldnot be compatible with stable transfection in COS cells. Five ug oflinear pHIV-Ad was co-transfected into COS cells in a 5 cm culture dishalong with a linearised pLTRneo vector specifying resistance to theantibiotic G418. Two days following transfection, the cells were split1:10 into medium containing 0.6 mg/mL G418 and resistant colonies wereallowed to develop. Individual colonies were isolated and expanded andRT activity was measured in 8 mL supernatants from each clone aspreviously described. RT activity was detected in the supernatants oftwo of the first 44 clones. These clones continued to produce highmolecular weight material associated with RT activity after continuouspassage in the presence of 0.6 mg/mL of G418.

Example 6

This Example illustrates the production of HIV-like particles in stablytransfected COS cells as demonstrated by Western blot analysis andsucrose density gradient centrifugation.

For Western blot analysis, a culture supernatant from stably transfectedCOS cells was layered over a 20% glycerol cushion and centrifuged at100,000×g for 90 minutes to pellet virus-like particles. The pellet wasresuspended in 1.5 uL of TNE for each mL of cell supernatant. Two twentyuL aliquots of the concentrated material were analyzed by Westernblotting with either a commercially available human HIV-1 specificantiserum (BioRad) or a cocktail of four commercial monoclonalantibodies specific for HIV-1 gp120, gp41, p24 and p17 as describedabove. These results demonstrated the presence of several bands withmolecular weights consistent with those of the following HIV-1 proteins:gp120, p55, gp41, p30, p23 and p17.

For sucrose density gradient centrifugation, HIV-like particles werepelleted from a 500 mL supernatant from stably transfected COS cells bysedimentation through a 20% glycerol cushion. The pellets wereresuspended in TNE and layered on a 20-60% sucrose density gradient andcentrifuged as described above. After centrifugation, the gradient wasfractionated and RT activity measured in each fraction. The results areshown in FIG. 6 and revealed the presence of a major peak of RT activitybanding at a density consistent with the formation of HIV-likeparticles.

Example 7

This Example illustrates the detection of budding particles in stablytransfected COS cells by electron microscopy.

For thin section analysis, stably transfected COS cells were scrapedfrom a culture flask, washed with growth medium, and pelleted bycentrifugation. Resuspended cells were fixed in 2.5% bufferedglutaraldehyde, followed by 1% buffered osmium tetroxide, dehydratedthrough alcohol and propylene oxide and embedded in an epon-aralditeepoxy resin mixture using standard techniques. Thin sections werestained with uranyl acetate and lead citrate and examined in a PhilipsEM3000 transmission electron microscope. The results demonstrated thepresence of budding, immature particles consistent with the productionof HIV-like particles.

Example 8

This Example illustrates the immunogenicity of HIV-like particlespurified from stably transfected COS cells by sucrose density gradientcentrifugation as described in Example 6.

Sucrose gradient fractions containing RT activity were diluted with tenvolumes of TNE and virus-like particles were concentrated bycentrifugation at 100,000×g for 90 minutes at 4° C. Purified virus-likeparticles were quantified as a function of their p24 content usingcommercial HIV-1 p24 assay kits (Abbott Laboratories and CoulterImmunology).

Six to eight week-old female mice, (C57BL/6×C₃ H)F1 (Charles River,Montreal) were immunized with HIV-1 virus-like particles and serumsamples were assayed for HIV-1 specific antibodies. Each mouse receivedtwo subcutaneous injections equivalent to 6 ug of p24 antigen perinjection at a three week interval. The primary and boostingimmunizations were performed in complete Freund's adjuvant andincomplete Freund's adjuvant, respectively. Nine days after the secondimmunization, sera were collected and heat-inactivated at 56° C. for 30minutes. HIV-1 specific antibodies were detected by enzyme-immunoassays(EIAs). EIA plates (Maxisorp, Nunc) were coated with recombinant (r)antigens: rgp120 (American Biotech. Inc.), rgp160 (Repligen), rgp41(DuPont), or rp24 (DuPont) in phosphate buffered saline (PBS, pH 7.0) at0.4 ug per well. Adsorption of the antigens was allowed to proceedovernight at 4° C. Unbound antigen was aspirated and the plates wereblocked with 300 uL per well of 2% skim milk powder (Carnation) in PBSfor two hours at room temperature. The plates were then washed threetimes with PBS containing 0.025% Tween 20 (BioRad). Serum samples wereserially diluted in PBS and added to individual wells for one hour atroom temperature. The plates were then washed three times with PBS/Tween20 as described above. A goat anti-mouse-IgG antibody conjugated tohorseradish peroxidase (Jackson Laboratories) was diluted 1 in 5000 withPBS and added for one hour at room temperature. After washing withPBS/Tween 20, 100 uL of a tetra-methylbenzidine substrate solutiondiluted 1 in 10 with peroxide reagent as described by the manufacturer(ADI Diagnostics) was added to each well for 10-15 minutes at roomtemperature. One hundred uL of 1N H₂ SO₄ was added to stop the reactionsand the plates were read in an EIA plate reader at 450 nm.

The results shown in FIG. 7 demonstrate significant IgG antibodyresponses against recombinant gp120, gp41, and p24 proteins. Thepresence of IgG antibodies against a major synthetic HIV-1neutralization epitope encompassing residues 311 to 320 of gp120 and asynthetic gp41 B-cell epitope (Residues 727 to 751) was detected inpeptide-specific EIA's using microtitre plates coated with syntheticpeptides produced by solid-phase chemical synthesis on an ABI Model 430Apeptide synthesizer. Murine antisera also demonstrated HIV-1 specificneutralization activity for HIV-1 strains LAV-1_(BRU) and HTLV-III_(MN)in a standard tissue culture syncytial inhibition assay at a serumdilution of 1/10. These results indicate that HIV-1-like particles havethe capacity to induce cross-reactive immune responses against differentviral isolates. Further evidence of antibody cross-reactivity wasobtained with sera from animals boosted a second time with virus-likeparticles at 1.5 ug of p24 core antigen per dose. The IgG responsesspecific for various HIV recombinant antigens are shown in FIGS. 8 and 9and demonstrate not only potent immune responses to HIV-1 gp120 andgp160, but also significant reactivity with HIV-2 gp120 as well. Thesedata indicate that HIV-like particles may prove to be an efficientimmunogen for the induction of cross-reactive immune responses.

Example 9

This Example illustrates the inducible expression of HIV-like particlesfollowing transfection of an inducible expression vector into Verocells.

FIG. 10 shows a map of the HIV expression vector pMT-HIV which issimilar to previous expression vectors but contains the humanmetallothionein IIa promoter from nucleotide positions -742 to +59,placed directly upstream of the HIV-1 coding sequences which begin atnucleotide position 678. This plasmid has been deposited with theAmerican Type Culture Collection on Oct. 12, 1990 (#40912). Afterco-tansfection of pMT-HIV into Vero cells with the G418 resistancemarker, numerous G418 resistant colonies were isolated and tested for RTproduction in response to cadmium chloride (CdCl₂) addition. Individualclones were grown to confluence in 9 cm culture dishes and treated with5 uM CdCl₂ for twenty four hours, after which a standard assay for RTactivity was performed on high molecular weight material pelleted from 8mL culture supernatants as previously described in Example 2. Theseresults demonstrated the production of significant levels of RT activityin at least 30% of the clones. RT activity in the absence of inductionwas detectable in the supernatants of only a few Vero cell clones. Thefollowing Table II shows the levels of p24 antigen production detectedin the culture supernatants of six clones in the presence and absence ofCdCl₂. p24 levels were measured using a commercial HIV-1 p24 assay kitand the results demonstrated a very high expression level for one clone(clone 11) and high induction ratios for all six clones.

                  TABLE II                                                        ______________________________________                                        p24 Production in Induced and Non-Induced                                     Vero Cell Clones                                                                                        Induced                                             Clone Number                                                                            Non-Induced (ug/L)                                                                            (ug/L)   Ratio                                      ______________________________________                                         7        <.01            16       >1600                                      10        <.01            33       >3300                                      11        .41             600       1463                                      30        .02             33        1650                                      62        .02             112       5600                                      76        <.01            74       >7400                                      ______________________________________                                    

Example 10

This Example illustrates the production of mature HIV-like particles inVero cell clone 11 which was stably transfected with the inducibleexpression vector pMT-HIV.

Subconfluent cells grown in a 75 cm² culture flask were treated with 5uM CdCl₂ for twenty four hours, then scraped from the flask andprocessed for thin section electron microscopy as described in Example7. Electron micrographs showed numerous mature and immature virus-likeparticles budding from the plasma membrane of the transfected cells.These levels of particle formation were consistent with the high levelsof p24 and RT production observed for this clone.

Example 11

This Example illustrates the continuous production of RT activity andp24 core antigen in a single flask of Vero cell clone II over an eightday period of continuous induction with CdCl₂.

Clone 11 cells were grown to confluence in a 75 cm² culture flask andwere then treated with 5 uM CdCl₂ for twenty four hours after which allof the medium was harvested and fresh medium containing CdCl₂ added.This process was repeated every twenty four hours for eight days and thelevels of RT and p24 production in each twenty four hour culturesupernatant are shown in FIG. 11. The levels of production of p24 and RTactivity correlated extremely well and peaked five days after the cellsreached confluence.

Example 12

This Example illustrates the feasibility of producing HIV-like particleswith a genetically modified envelope glycoprotein.

It is possible that structural alterations of gp120 may result in areduction of its binding affinity for gp41, and therefore in itsshedding from the surface of the particles. Mutations preventing theproteolytic processing of the gp160 envelope glycoprotein shouldcircumvent this problem and result in the expression of uncleavedglycoprotein precursors stably associated with virus-like particles.Codon position 455 in the LAV-1_(BRU) gp160 envelope glycoprotein geneencodes an arginine residue which is the site of proteolytic cleavage inthe gp160 envelope glycoprotein. This codon was mutated to encode athreonine residue using a commercial M13 phage-based site-directedmutagenesis kit (Amersham) and an appropriate synthetic mutagenicoligonucleotide complementary to the regions flanking this codon. Thismutation was created in a cloned subfragment of the envelopeglycoprotein gene which was subsequently used to replace thecorresponding region in the pHIV-SV particle expression plasmid.Transfection of this modified plasmid into COS cells resulted in theexpression of high molecular weight virus-like material in associationwith the gp160 envelope glycoprotein precursor as detected by Westernblot analysis. The fact that gp120 was not detected was consistent withthe disruption of proteolytic processing. That the gp160 envelopeprotein remains associated with virus-like particles was demonstrated bybanding particles produced in a transient transfection experiment bysucrose density gradient centrifugation. Western blot analysisdemonstrated that the gp160 precursor protein was found only in gradientfractions containing RT activity and not in heavier or lighterfractions.

The feasibility of modifying the structure of the gp120 envelopeglycoprotein was further demonstrated by inserting two pairs ofsynthetic oligonucleotides into the BglII site at nucleotide position7008 in the LAV-1_(BRU) DNA sequence. Insertion of theseoligonucleotides resulted in a modified coding sequence in which theinserted element encoded the HTLV-III_(MN) V3 neutralization epitopeencompassing amino acid residues 306 to 329 of the viral envelopeglycoprotein. The resultant sequence encoded the complete LAV-1_(BRU)envelope glycoprotein with an MN strain V3 loop insertion at amino acidposition 272. The resulting plasmid is termed pV3Bg and has beendeposited with the American Type Culture Collection on Oct. 12, 1990(#40910). The oligonucleotides used in the modification were as follows:##STR1## Western blot analysis of pellets obtained by centrifugation oftransfected cell supernatants through a 20% glycerol cushiondemonstrated the production of particulate material containing gp120 ata level similar to that observed with the unmodified expression vector.These data demonstrate that the insertion of 24 additional aminoacidresidues at amino acid position 272 of the LAV-1 envelope did notdisrupt its targeting to the viral membrane, the processing of theenvelope glycoprotein precursor, or its non-covalent association withthe gp41 transmembrane glycoprotein. It is therefore likely thatadditional gp120 alterations might be tolerated and, thus, allow forfurther modulation of the immunogenicity of the envelope glycoprotein.

Example 13

This Example illustrates the feasibility of creating a mutation in anHIV-1 sequence element which is required for infectivity but dispensablefor virus-like particle production.

To demonstrate the feasibility of producing HIV-like particles using anexpression construct containing an additional mutation in a geneticelement required for infectivity, an expression plasmid containing adeletion of the RNA packaging signal was created. This signal has beenshown to lie between the first splice donor at nucleotide position 744and the initiation codon for Gag at nucleotide position 790 and to becritical for HIV RNA packaging and infectivity. This region was deletedfrom a clone containing an 8.9 kb SacI-SacI fragment of LAV-1_(BRU)(nucleotide positions 678-9619) by removing a 114 bp SacI-HqaIrestriction fragment encompassing nucleotide positions 678 to 791. Thedeleted fragment was replaced with a double-stranded synthetic fragmentcontaining a deletion of 26 bp from nucleotide positions 753 to 777. Themodified SacI-SacI fragment was then used as a source of DNA to isolatethe SacI-XhoI fragment (nucleotide positions 678 to 8944) forconstruction of an HIV expression vector utilizing the adenovirus majorlate promoter similar to pHIV-Ad described in Example 5. The newplasmid, termed pHIV-Ad-d26 was transfected into COS cells as describedin Example 5 and G418 resistant clones constitutively expressing RTactivity were isolated. Virus-like particles were pelleted from 200 mLof culture supernatants from two COS cell clones stably transfected withthe original and "RNA packaging-deleted" expression constructs,respectively. FIG. 12 shows a comparison of the sucrose density gradientcentrifugation profiles for the virus-like particles derived from theseexpression constructs. Virus-like particles derived from the constructcontaining the packaging deletion were not significantly different fromthose derived from the original expression construct.

Example 14

This Example illustrates the production of non-infectious SIV-likeparticles.

To demonstrate the applicability of this invention to the production ofvirus-like particles derived from a retrovirus other than HIV, an SIVexpression vector containing a DNA fragment from the provirus ofSIV_(mac239) was constructed. Although the nucleotide sequence of theSIV_(mac239) provirus has yet to be published, comparison of itsrestriction map with that of SIV_(mac142) which has been sequencedrevealed a number of similarities. The NarI restriction site atnucleotide position 835 of SIV_(mac142) and the two SstI restrictionsites at positions 5756 and 9236 are conserved in the SIV_(mac239)provirus. Therefore, two fragments from the SIV_(mac239) provirusconsisting of the 4.9 kb NarI-SstI and the 3.4 kb SstI-SstI fragmentswere isolated from the cloned SIV_(mac239) provirus and inserted into anexpression vector utilizing the SV40 virus early promoter as describedin Example for LAV-1_(BRU). Transfection of this plasmid, termedpSIV-SV, into COS cells resulted in the transient expression of RTactivity as described for LAV-1_(BRU) ' also in Example 2. Theco-transfection of Vero cells with pSIV-SV and a G418 resistance markerresulted in the isolation of drug resistant clones constitutivelyexpressing RT activity as described in Example 5 for the transfection ofpHIV-Ad into COS cells.

To provide further evidence for the production of SIV-like particles,culture supernatants were collected from Vero cells stably transfectedwith pSIV-SV and virus-like particles were pelleted by centrifugation at100,000×g. Pelleted material was analyzed by SDS-PAGE and Westernblotting using a monkey SIV-specific antiserum. Analysis of the datarevealed the presence of several bands with molecular weights consistentwith the expression of the major SIV antigens. These bands were notdetected in the culture fluid of non-transfected Vero cells.

Example 15

This Example illustrates the feasibility of expressing HIV-likeparticles using an expression vector containing a deletion of theprovirus genome which encodes proteins required for viral infectivitybut which are dispensable for particle assembly.

The pMT-HIV expression vector described in Example 9 was modified tocontain a deletion of the 26 nucleotides in the RNA packaging region asdescribed in Example 13. A further deletion of HIV coding sequenceswhich encode the Vif and Integrase genes was also performed. The Vif andIntegrase genes are required for viral infectivity but are dispensablefor particle assembly. This second deletion was accomplished by removinga 746 bp fragment between the BspmI restriction sites at nucleotidepositions 4345 and 5091 of the LAV-1_(BRU) genome. The resultantexpression plasmid, termed pMT-HIV-dVI-d26, has been deposited with theAmerican Type Culture Collection on Oct. 12, 1990 (#40911). Transfectionof pMT-HIV-dVI-d26 into COS cells in a transient expression assayresulted in the expression of significant quantities of p24 antigen inthe culture supernatant but no RT activity was detected. The absence ofRT activity indicates that the neighbouring integrase mutations may haveaffected RT protein processing. Evidence for the assembly of virus-likeparticles was obtained from a Western blot analysis of material pelletedfrom the culture supernatant of transfected cells by high speedcentrifugation through a 20% glycerol cushion which demonstrated thepresence of gp120 in significant quantities. Production of p24 and gp120was not detected in the culture supernatant of non-transfected cells.

Example 16

This Example illustrates the production of HIV-like particles which aredeficient in the gp120 envelope glycoprotein due to a deletion of gp120coding sequences in a modified expression vector.

The HIV expression vector pHIV-SV was modified so as to encode theproduction of virus-like particles lacking the gp120 envelopeglycoprotein. This was accomplished by deleting a fragment between theKpnI site at nucleotide position 6379 and the BglII site at nucleotideposition 7668. The remaining restriction ends were treated with KlenowDNA polymerase to remove the 3' overhang at the KpnI end and to fill inthe 5' overhang at the BglII end. The blunt ends were ligated to a BglIIlinker (Pharmacia, 5'-pd[CAGATCTG]-3') which was then cleaved with BglIIand the sticky ends were ligated together. This ligation restored whatremained of the gp120 reading frame to allow for the later insertion ofheterologous coding sequences. This plasmid, termed pBL-HIV-dgp120-6,has been deposited with the American Type Culture Collection on Oct. 12,1990 (#40913).

Transfection of pBL-HIV-gdp120-6 into COS cells in transient expressionassays resulted in the expression of high molecular weight materialcontaining RT activity. Western blot analysis of the pellet obtained byhigh speed centrifugation of the culture supernatant through a 20%glycerol cushion revealed the expression of the p24 and p17 gag proteinsbut not of gp120. In a control experiment where the original pHIV-SVexpression plasmid was transfected, significant quantities of HIV-1gp120 were detected in the pelleted fractions.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention providesenetically-engineered non-infectious and immunogenic retrovirus-likeparticles which are candidates for vaccines against the respectiveretroviruses, such as AIDS and procedures for producing the same.Modification are possible within the scope of this invention.

What we claim is:
 1. A genetically-engineered, non-infectious,non-replicating and immunogenic HIV retrovirus-like particle, producedby:incorporating into an expression vector a DNA molecule comprising theHIV genome devoid of long terminal repeats, introducing the expressionvector into mammalian cells, and expressing said DNA molecule in saidmammalian cells to produce said HIV retrovirus-like particle.
 2. Theparticle of claim 1 which is deficient in primer binding site.
 3. Theparticle of claim 1 which is deficient in Integrase and Vif proteins. 4.The particle of claim 1 wherein a V3 neutralizing epitope from aheterologous HIV isolate is inserted into a Bgl II site of the envelopeglycoprotein.
 5. A method for obtaining a non-infectious,non-replicating, immunogenio HIV retrovirus-like particle by geneticengineering, which comprises:incorporating into an expression vector aDNA molecule comprising the HIV genome devoid of long terminal repeats,introducing the expression vector into mammalian cells, and expressingsaid DNA molecule in said mammalian cells to produce HIV retrovirus-likeparticles.
 6. The method of claim 5 wherein said expression vector isplasmid pHIV-SV.
 7. The method of claim 5 wherein said expression vectoris plasmid pHIV-CHO-SV.
 8. The method of claim 5 wherein said expressionvector is plasmid pHIV-Ad.
 9. The method of claim 5 wherein saidexpression vector is plasmid pMT-HIV.
 10. The method of claim 5 whereinsaid expression vector is plasmid pV3Bg.
 11. The method of claim 6wherein said HIV genome further is deficient in primer binding site. 12.The method of claim 11 wherein said HIV genome further is deficient ingenomic elements coding for Intergrass and Vif.
 13. The method of claim5 wherein said DNA molecule incorporated into the expression vector isprovided by a DNA molecule containing the characteristic geneticelements present in the SacI to XhoI fragment spanning nucleotides 678to 8944 of the genome of HIV-1 BRU isolate.